The human malaria parasite is endemic in 87 countries putting 2.5 billion people in the poorest nations of the tropics at risk for the disease. Despite intensive efforts to control malaria through combination drug therapy and insect control programs, malaria remains one of the largest global health problems. Unfortunately wide-spread drug resistance to almost every known anti-malarial agent has compromised the effectiveness of malaria control programs. Pyrimidine biosynthesis provides a significant opportunity for the development of new chemotherapeutic agents against the malaria parasite. Plasmodium species rely exclusively on de novo pyrimidine biosynthesis to provide precursors for DNA and RNA synthesis, while mammalian cells have salvage pathways that provide an alternative route to these essential metabolites. Dihydroorotate dehydrogenase (DHODH) catalyzes the fourth step in de novo pyrimidine biosynthesis and it has been shown to be essential to the parasite by genetic studies. DHODH is a demonstrated "druggable" target as inhibitors of the enzyme are approved for the treatment of rheumatoid arthritis. We identified a number of chemical scaffolds that are potent and species selective inhibitors of P. falciaprum DHODH by high throughput screening (HTS). A triazolopyrimidine-based series emerged from this screen that has potent activity against malaria parasites in vitro, and which is able to suppress parasites in a mouse model of the disease. We solved the X-ray structures of PfDHODH bound to inhibitors from this series and these data showed that species selectivity of binding results from species specific inhibitor binding modes that arise from amino acid differences between the human and malarial enzymes in the inhibitor binding site. These data led to the validation of DHODH as a target for new drug development in malaria and this compound series is forming the basis of a lead optimization program in a separately funded application. The objectives of this application are to perform Hit-lead-optimization of additional chemical scaffolds discovered in our HTS screen for DHODH inhibitors, with the goal to identify 1-2 additional compounds that are ready for full scale lead optimization programs as backups to our current triazolopyrimidine series. Secondly the application is focused on providing insight into the biological effects of PfDHODH inhibitors on the malaria parasite, through study of mechanism of action and study of the mechanisms of resistance. The Hit-to-lead optimization of additional inhibitor classes will be informed by activity assays (both on DHODH and in cell based assays) and X-ray crystallography. Mechanism of action studies will be conducted using transgenic parasites that overcome the block in DHODH and by metabolite analysis using LC/MS approaches. Finally parasites resistant to the different classes of DHODH inhibitors will be studied to understand both the frequency and mechanism by which cells become resistant to these inhibitors. In combination, these studies will greatly facilitate the discovery of much needed new drugs for the treatment of malaria